Reactive Oxygen Species-generating Mitochondrial DNA Mutation Up-regulates Hypoxia-inducible Factor-1α Gene Transcription via Phosphatidylinositol 3-Kinase-Akt/Protein Kinase C/Histone Deacetylase Pathway
Reactive Oxygen Species-generating Mitochondrial DNA Mutation Up-regulates Hypoxia-inducible Factor-1α Gene Transcription via Phosphatidylinositol 3-Kinase-Akt/Protein Kinase C/Histone Deacetylase Pathway
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DOI：
10.1074/jbc.m109.054221
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发表时间：
2009-11-27
影响因子：
4.8
通讯作者：
Takenaga, Keizo
中科院分区：
文献类型：
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作者：
Koshikawa, Nobuko;Hayashi, Jun-Ichi;Takenaga, Keizo
与低转移性 P29 细胞相比，Lewis 肺癌来源的高转移性 A11 细胞持续过度表达缺氧诱导因子 (HIF)-1 α mRNA。由于 A11 细胞的线粒体 NADH 脱氢酶亚基 6 (ND6) 基因仅具有 G13997A 突变，因此我们在此研究了 ND6 突变与 HIF-1 α 转录激活之间的因果关系，并研究了其潜在机制。使用 A11 和 P29 细胞之间的跨线粒体杂种，我们发现 ND6 突变直接参与 HIF-1 α mRNA 过度表达。 ND6 突变对 HIF-1 α 转录的刺激是由活性氧 (ROS) 的过量产生以及随后磷脂酰肌醇 3-激酶 (PI3K)-Akt 和蛋白激酶 C (PKC) 信号通路的激活介导的。 Sp1 抑制剂光神霉素 A 消除了 HIF-1 α 转录的上调，但荧光素酶报告基因和染色质免疫沉淀分析表明，Sp1 对于 A11 细胞中 HIF-1 α mRNA 过表达来说是必要的，但还不够。另一方面，曲古抑菌素 A（一种组蛋白脱乙酰酶 (HDAC) 抑制剂）可显着抑制 A11 细胞中的 HIF-1 α 转录。据此，A11细胞中的HDAC活性较高，但P29细胞和用ROS清除剂依布硒啉、PI3K抑制剂LY294002和PKC抑制剂Ro31-8220处理的A11细胞中的HDAC活性较低。这些结果表明，产生 ROS 的 ND6 突变通过 PI3K-Akt/PKC/HDAC 途径增加 HIF-1 α 转录，导致缺氧肿瘤细胞中 HIF-1 α 蛋白积累。
Lewis lung carcinoma-derived high metastatic A11 cells constitutively overexpress hypoxia-inducible factor (HIF)-1 alpha mRNA compared with low metastatic P29 cells. Because A11 cells exclusively possess a G13997A mutation in the mitochondrial NADH dehydrogenase subunit 6 (ND6) gene, we addressed here a causal relationship between the ND6 mutation and the activation of HIF-1 alpha transcription, and we investigated the potential mechanism. Using trans-mitochondrial cybrids between A11 and P29 cells, we found that the ND6 mutation was directly involved in HIF-1 alpha mRNA overexpression. Stimulation of HIF-1 alpha transcription by the ND6 mutation was mediated by overproduction of reactive oxygen species (ROS) and subsequent activation of phosphatidylinositol 3-kinase (PI3K)-Akt and protein kinase C (PKC) signaling pathways. The up-regulation of HIF-1 alpha transcription was abolished by mithramycin A, an Sp1 inhibitor, but luciferase reporter and chromatin immunoprecipitation assays indicated that Sp1 was necessary but not sufficient for HIF-1 alpha mRNA overexpression in A11 cells. On the other hand, trichostatin A, a histone deacetylase (HDAC) inhibitor, markedly suppressed HIF-1 alpha transcription in A11 cells. In accordance with this, HDAC activity was high in A11 cells but low in P29 cells and in A11 cells treated with the ROS scavenger ebselene, the PI3K inhibitor LY294002, and the PKC inhibitor Ro31-8220. These results suggest that the ROS-generating ND6 mutation increases HIF-1 alpha transcription via the PI3K-Akt/PKC/HDAC pathway, leading to HIF-1 alpha protein accumulation in hypoxic tumor cells.